@MilesAfton Hi. Hmm honestly we don’t really know what is the exact limit for the number of layers, if this number is stored inside a container with a type of integer numbers the limit could be about 35 thousand, but this is just speculation.
The size of a project file is only limited to the size of your RAM. I have seen, and can open files with 1 gigabyte of size, but I don’t recommend this, you will need a powerful computer to open the file and work with it. It’s always better to work smaller files and edit them together with a dedicated video editor.
If you can’t open your file, send me a private message with a copy and i’ll try to open it and extract the information, but honestly please work by using one Pencil2D file per every shot of your animation. It’s the best way to avoid potential issues in case of file corruption.